Gene expression profiling of endocycling cells vs mitotic cells

In this study we used expression microarrays as an entry point to gain further insights into the differences between endocycling cells and mitotic cycling in tissues of the Drosophila larva. Gene expression analyses were performed on tissues hand dissected from yw67c2 early 3rd instar larval tissues collected 70-75hr after egg laying. Total RNA was isolated using RNeasy Micro Kit (QIAGEN Valencia, CA 91355), and reverse transcribed and fluorescently labeled with Cy3 or Cy5 (Amersham) using Amino Allyl MessageAmp™ II aRNA Amplification Kit (Ambion, Austin, TX 78744), according to the manufacturer's instructions. This was used to probe the DGRC-2 array, a Drosophila oligonucleotide array created by the Drosophila Genomics Resource Center (DGRC) which represents ~93% of annotated Drosophila genes from genome release 4.1 (Bogart et al., 2006). Two color microarray hybridizations were repeated with material from independent dissections representing two (fat body versus brain) and three (salivary gland versus brain) independent biological replicates. Slides were scanned by GenePix 4100A (Axon Instruments, Union City, CA) and the data was extracted with Axon GenePix Pro 5 image analysis software.